The close relationship between increased resistance to stress and extended life span has been extensively demonstrated in invertebrates such as yeast, the fruit fly, and C. elegans. Several long-lived mouse mutants are also resistant to multiple forms of stress, including oxidants, UV, and heavy metals. This relationship holds equally true for the exceptionally long-lived naked mole rat. Selection for stress-resistance has been successfully employed as a surrogate marker to identify long-lived mutants in lower organisms. Based on these findings, we have pioneered novel approaches to apply methodology usually only available in microbial systems: using stress resistance as a selectable surrogate marker to generate long-lived mutants in the mouse. We have developed novel strategies to allow mutagenesis and mass selection for stress resistance in mouse embryonic stem (ES) cells and have coupled these to a method wherein these ES cells maintain both the stress resistant phenotype and pluripotency, thus allowing us to generate mouse mutants that are stress- resistant. These new mouse models allow us to critically test the hypothesis that increased cellular stress-resistance slows aging and improves life and health span in mammals. These are valuable models and we have numerous collaborators awaiting access to these strains; letters from five labs are attached to this application. Since the procedure is not limite to previously identified genes and pathways, it allows for unbiased detection of novel genes affecting multi-stress resistance in mice. Several refinements are incorporated in this application allowing us to detect both dominant and recessive mutations and to rapidly identify the mutated gene(s). To carry out these novel high-throughput strategies we have constructed novel transposon vectors and will generate a genome-wide library of multi-stress resistant mutant mice. We screen for mutant ES cell clones that are resistant to paraquat and we screen these for multi-stress resistance, levels of reactive oxygen species under stress, pluripotency, expression levels of Nrf2 and other antioxidant genes, and their capability of maintaining stress-resistance after differentiation into other cell types. The gene-trapped alleles conferring stress-resistance are easily identified and verified. A collection of stress- resistant ES cell clones are thus generated and will be deposited in the public cell repository as shared resources. We will generate strains of mouse mutants from these cells for in vivo studies of aging in the mouse. The impact of increased cellular stress-resistance on life span, gross development, general behavior, end-of-life pathology, and age-related diseases will be examined. Importantly, the causative gene(s) for life span extension will also be validated; these genes and pathways are potential targets for pharmacological intervention to slow aging and ameliorate multiple diseases of aging in humans. In summary, the success of these studies will identify new genes implicated in stress-resistance and perhaps modulating mammalian life span and health. We anticipate that the insights gained from these studies will foster a variety of new directions for aging research.